Methods to culture circovirus

ABSTRACT

The present invention relates to methods for culturing circovirus and in particular, porcine circovirus. The present invention provides compositions and methods for culturing porcine circovirus in mammalian cells expressing mammalian adenovirus E1 functional protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationSerial No. 60/279,173, filed Mar. 27, 2001, hereby incorporated hereinin its entirety.

TECHNICAL FIELD

The present invention relates to the field of circovirus and providescompositions and methods for culturing circovirus, in particular porcinecircovirus. In particular, the present invention relates to methods forculturing porcine circovirus in mammalian cells expressing a mammalianadenovirus E1 gene function.

BACKGROUND ART

A family of viruses, named Circoviridae, found in a range of plant andanimal species and commonly referred to as circoviruses, arecharacterized as round, non-enveloped virions with mean diameters from17 to 23.5 nm containing circular, single-stranded deoxyribonucleic acid(ssDNA). The ssDNA genome of the circoviruses represent the smallestviral DNA replicons known. As disclosed in WO 99/45956, at least sixviruses have been identified as members of the family according to TheSixth Report of the International Committee for the Taxonomy of Viruses(Lukert, P. D. et al. 1995, The Circoviridae, pp. 166-168. In F. A.Murphy, et al. (eds.) Virus Taxonomy, Sixth Report of the InternationalCommittee on Taxonomy of Viruses, Arch. Virol. 10 Suppl.).

Animal viruses included in the family are chicken anemia virus (CAV);beak and feather disease virus (BFDV); porcine circovirus (PCV); andpigeon circovirus. PCV was originally isolated in porcine kidney cellcultures. PCV replicates in the cell nucleus and produces largeintranuclear inclusion bodies. See Murphy et al. (1999, Circoviridae p.357-361, Veterinary Virology, 3rd ed. Academic Press, San Diego). Thereare currently two recognized types of PCV, PCV type 1 (PCV1) and PCVtype 2 (PCV2). PCV1, isolated as a persistent contaminant of thecontinuous porcine kidney cell line PK-15 (ATCC CCL31), does not causedetectable cytopathic effects in cell culture and fails to produceclinical disease in pigs after experimental infection (see Allan G.,1995, Vet. Microbiol. 44: 49-64; Tischer, I. et al., 1982, Nature295:64-66; and Tischer, I. et al., 1986, Arch. Virol. 91:271-276). PCV2,in contrast to PCV1, is closely associated with post weaningmultisystemic wasting syndrome (PMWS) in weanling pigs (see Allan G. etal., 1998, Europe. J. Vet. Diagn. Investig. 10:3-10; Ellis, J. et al.,1998, Can. Vet. J. 39:44-51 and Morozov, I. et al., 1998, J. Clin.Microbiol. 36:2535-2541). The nucleotide sequences for PCV1 aredisclosed in Mankertz, A., et al. (1997, J. Virol. 71:2562-2566) andMeehan, B. M., et al. (1997, J. Gen. Virol. 78:221-227) and thenucleotide sequences for PCV2 are disclosed in Hamel, A. L. et al.(1998, J. Virol. 72:5262-5267); Mankertz, A. et al. (2000, Virus Res.66:65-77) and Meehan, B. M. et al. (1998, J. Gen. Virol. 79:2171-2179).Strains of PCV2 are disclosed in WO 00/01409 and have been deposited atthe European Collection of Cell Cultures, Centre for AppliedMicrobiology & Research, Porton Down, Salisbury, Wiltshire SP4 OJG,United Kingdom and include: accession No. V97100219; accession No.V9700218; accession No. V97100217; accession No. V98011608; andaccession No. V98011609. WO 00/77216 also discloses PCV2.

Published studies to date on PCV2 used either tissue homogenate orcultured virus derived from field isolates. Tischer et al. (1987, ArchVirol. 96:39-57) report that porcine kidney cells are stimulated toentry to the S phase in the cell cycle by D-glucosamine treatment.However, the treatment must be performed with caution becauseD-glucosamine is toxic for cell culture (see, Allan et al., (2000). J.Vet. Diagn. Investigation. 12:3-14). There remains a need for methodsfor culturing circovirus, such as for example, PCV1 and PCV2, and othercircoviruses, such that pure circovirus is obtained. Such methods wouldbe advantageous, in particular for preparation of PCV2 antigens asvaccines directed against PMWS. The present invention addresses thatneed.

All patents and publications are hereby incorporated herein in theirentirety.

DISCLOSURE OF THE INVENTION

The present invention provides methods for culturing mammaliancircovirus comprising: a) obtaining mammalian cells expressing amammalian adenovirus E1 function, wherein said cells are permissive formammalian circovirus replication; b) introducing said mammaliancircovirus genome, or a portion thereof capable of replication, intosaid mammalian cells; and c) culturing said mammalian cells underconditions suitable for replication of said mammalian circovirus. Insome embodiments, the method further comprises recovering saidcircovirus from said cultured cells.

In some embodiments, the mammalian circovirus is porcine circovirus,such as for example, porcine circovirus 1 (PCV1) or porcine circovirus 2(PCV2). In yet additional embodiments, the porcine circovirus comprisesa chimeric nucleotide sequence. In other embodiments, the mammaliancells are of porcine origin. In yet other embodiments, the mammaliancells are porcine retina cells.

In other embodiments, the mammalian adenovirus E1 function is humanadenovirus E1 function. In yet other embodiments, the mammalianadenovirus E1 function is porcine adenovirus E1 function. In furtherembodiments, the E1 function is E1A and/or E1B function. In yet furtherembodiments, the mammalian cell expressing the mammalian E1 function isstably transformed with mammalian E1 gene sequences. In otherembodiments, the mammalian E1 gene sequence is heterologous to saidmammalian cell.

The present invention also provides recombinant mammalian cells thatexpress a mammalian adenovirus E1 function and comprise a mammaliancircovirus genome, or a portion thereof capable of replication, andwherein said cells are permissive for the replication of said mammaliancircovirus. In some embodiments, the mammalian circovirus is porcinecircovirus, such as for example, porcine circovirus 1 (PCV1) or porcinecircovirus 2 (PCV2). In yet additional embodiments, the porcinecircovirus comprises a chimeric nucleotide sequence. In someembodiments, the adenovirus E1 function is human adenovirus E1 function.In other embodiments, the E1 function is porcine adenovirus E1 function.In other embodiments, the mammalian cell is of porcine origin. Infurther embodiments, the mammalian cell is a porcine retinal cell. Inyet further embodiments, the mammalian cell expressing the mammalian E1function is stably transformed with mammalian adenovirus E1 genesequences. In other embodiments, the mammalian E1 gene sequence isheterologous to said mammalian cell.

The present invention also provides methods of preparing a recombinantmammalian cell expressing a mammalian adenovirus E1 function andcomprising a mammalian circovirus genome comprising the steps of, a)obtaining a mammalian cell expressing a mammalian adenovirus E1function; and b) introducing said mammalian circovirus genome, or aportion thereof capable of replication, into said mammalian cell. Inadditional embodiments, the method comprises the additional step ofculturing the recombinant mammalian cell under conditions suitable forthe replication of said mammalian circovirus. In further embodiments,the method comprises recovering said circovirus from said culturedcells. In some embodiments, the mammalian circovirus is porcinecircovirus, such as for example, porcine circovirus 1 (PCV1) or porcinecircovirus 2 (PCV2). In yet additional embodiments, the porcinecircovirus comprises a chimeric nucleotide sequence. In furtherembodiments, the mammalian cells are of porcine origin. In yet furtherembodiments, the mammalian cells are porcine retina cells. In additionalembodiments, the adenovirus E1 function is human adenovirus E1 functionor porcine adenovirus E1 function. In yet further embodiments, themammalian cell expressing the mammalian adenovirus E1 function is stablytransformed with mammalian adenovirus E1 gene sequences. In otherembodiments, the mammalian E1 gene sequence is heterologous to saidmammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B provide the characterization and titration of PCV2 virusgenerated by DNA transfection and extraction from infected VIDO R1 cellsby Hirt's method. (A) PCR using PCV2-specific primers and DNA fromPCV2-infected (lane 1) and mock-infected (lane 2) cells. A plasmidcontaining PCV2 genome was used as a control (lane 3). The 1-kb DNAladder from GIBCO BRL was loaded in lane M. (B) Viral DNA fromPCV2-infected (lanes 1, 3, and 5) and mock-infected (lanes 2, 4, and 6)cells were digested with NcoI and StuI (lanes 1 and 2), EcoRI and StuI(lanes 3 and 4), and EcoRI and EcoRV (lanes 5 and 6). The 1-kb-plus DNAladder from GIBCO BRL was loaded in lane M.

FIGS. 2A-2B depict titration of PCV2 by immunoperoxidase staining. At 72h.p.i., mock-(A) or PCV2-(B) infected VIDO R1 cells were incubated withrabbit anti-ORF2 polyclonal antibody and biotinylated secondaryantibody. After application of an avidin and biotinylated horseradishperoxidase complex, the monolayer was developed by diaminobenzidinetetrahydrochloride (DAB). One dark cell resulted from one virus particleinfection.

FIGS. 3A-3C show the nucleotide sequence (A) (SEQ ID NO:1) and aminoacid sequence for ORF 1 (B) (SEQ ID NO:2) and ORF 2(C) (SEQ ID NO:3) ofporcine circovirus 2 (PCV2) as described in Genbank accession numberAF086834.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to compositions and methods for culturingmammalian circovirus, in particular porcine circovirus. The presentinvention is based on the finding that a porcine cell expressing humanE1 function was able to be transfected with a PCV2 virus genome andgenerated PCV2 virus with a high virus titer. VIDO R1 cell line,deposited with the ATCC and having ATCC accession number PTA-155, is aporcine retina cell line transformed with human adenovirus-5 (HAV5) E1,that has been shown to induce the S phase of the cell cycle andtransactivate transcription. See, Shenk, T. (1996). “Adenoviridae: theviruses and their replication” In Fields Virology. 3^(rd) ed. B. N.Fields, D. M. Knipe and P. M. Howley (ed.) Lippincott-Raven Publishers,Philadelphia, N.Y., pp. 2111-2148. As described herein in Example 3,VIDO R1 cells, were transfected with a PCV2 genome and generated virusat 2×10⁷ IU/ml.

The practice of the present invention employs, unless otherwiseindicated, conventional microbiology, immunology, virology, molecularbiology, and recombinant DNA techniques which are within the skill ofthe art. These techniques are fully explained in the literature. See,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual (1982);DNA Cloning: A Practical Approach, vols. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed. (1984)); Nucleic AcidHybridization (B. Hames & S. Higgins, eds. (1985)); Transcription andTranslation (B. Hames & S. Higgins, eds. (1984)); Animal Cell Culture(R. Freshney, ed. (1986)); Perbal, A Practical Guide to MolecularCloning (1984); Ausubel, et al., Current Protocols In Molecular Biology,John Wiley & Sons (1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995,1996); and Sambrook et al., Molecular Cloning: A Laboratory Manual(2^(nd) Edition); vols. I, II & III (1989).

Circoviridae, a family of viruses having round, non-enveloped virionswith mean diameters from 17 to 23.5 nm containing circular,single-stranded DNA (ssDNA), are described in The Sixth Report of theInternational Committee for the Taxonomy of Viruses, supra. Members ofthe group include the porcine circoviruses, PCV1 and PCV2. Some of thePCVs are known to be pathogenic, such as PCV2, associated with PMWS.

Nucleotide sequences for PCV1 are provided in Mankertz, A., et al.,1997, J. Virol. 71:2562-2566 and Meehan, B. M. et al., 1997, J. Gen.Virol. 78:221-227. Nucleotide sequences for PCV2 are provided in Hamel,A. L. et al., 1998, J. Virol. 72:5262-5267; Mankertz, A. et al., 2000,Virus Res. 66:65-77 and Meehan, B. M. et al., 1998, J. Gen. Virol.79:2171-2179. Representative strains of PCV2 have been deposited withthe European Collection of Cell Cultures, Centre for AppliedMicrobiology & Research, Porton Down, Salisbury, Wiltshire SP4 OJG,United Kingdom and include accession No. V97100219; accession No.V9700218; accession No. V97100217; accession No. V98011608; andaccession No. V98011609. WO 00/77216 also discloses PCV2. PCV2nucleotide sequences have also been published in Hamel et al., (1998),J. Virol. vol. 72, 6:5262-5267 (GenBank AF027217) and in Morozov et al.,(1998), J. Clinical Microb. vol. 36, 9:2535-2541, as well as GenBankAF086834; AF086835; and AF086836. Comparison of the published nucleotidesequences for PCV1 and PCV2 reveals a <80% identity, although thegenomic organization is similar, especially in the arrangement of thetwo largest open reading frames (ORFs) with a putative origin of DNAreplication.

The present invention encompasses methods of culturing mammaliancircovirus and in particular, porcine circovirus (PCV). The presentinvention encompasses methods of culturing PCV comprising the PCVnucleotide sequences disclosed herein or known in the art, or ORFsthereof, or portions thereof that are capable of replication. Thepresent invention also encompasses methods of culturing PCV having PCVnucleotide sequences differing through the degeneracy of the geneticcode to those disclosed herein or known in the art, or ORFs thereof, orportions thereof capable of replication. The present invention furtherencompasses methods of culturing PCV comprising PCV nucleotide sequencevariations which do not change the functionality or strain specificityof the nucleotide sequence, or ORFs thereof, or portions thereof capableof replication. The present invention also encompasses methods ofculturing PCV comprising PCV nucleotide sequences capable of hybridizingto those sequences disclosed herein under conditions of intermediate tohigh stringency, and methods of culturing PCV comprising mutations ofthe PCV nucleotide sequence disclosed herein or known in the art, suchas deletions or point mutations, or ORFs thereof, or portions thereof,capable of replication. The present invention also encompasses methodsof culturing PCV comprising heterologous nucleotide sequences. Thepresent invention encompasses methods of culturing PCV that comprisechimeric circovirus nucleotide sequences, such as, for example,nucleotide sequences from porcine circovirus in fusion with nucleotidesequences from other pathogenic viruses, such as a pathogenic porcinevirus, including parvovirus.

As used herein, a heterologous nucleotide sequence, with respect to acircovirus or mammalian cell, is one which is not normally associatedwith the circovirus sequences as part of the circovirus genome or onewhich is not normally associated with the mammalian cell, respectively.Heterologous nucleotide sequences include synthetic sequences.Hybridization reactions can be performed under conditions of different“stringency”. Conditions that increase stringency of a hybridizationreaction are widely known and published in the art. See, for example,Sambrook et al. (1989) at page 7.52. Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC,6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionizedwater. An exemplary set of stringent hybridization conditions is 68° C.and 0.1×SSC.

The PCV genomes encode several polypeptide sequences, ranging inapproximate size from 8 to 35 kD. It is deemed routine to determine openreading frames (ORFs) for porcine circoviruses using standard softwaresuch as for example, MacVector® (Oxford Molecular Group Inc., MD 21030).The largest ORF, ORF1, of the two types of PCV shows only minorvariation with an identity of 85% (as measured by the clustal program)and has been demonstrated to be the Rep protein in PCV1 (Mankertz, A.,et al., 1998, J. Gen. Virol. 79:381-384). Without wanting to be bound bytheory, a higher rate of variation displayed in the ORF2 sequences ofPCV1 and PCV2 (identity about 65%) would suggest that type-specificfeatures of PCV might be determined by the respective ORF2 protein.Several PCV type-specific epitopes have been mapped on PCV2 ORF2sequences. See Mahe, D. et al., 2000, J. Gen. Virol. 81:1815-24. Inanother recent study, PCV2 ORF2 has been identified as a majorstructural protein that can form viral capsid-like particles in insectcells infected with ORF2 expressing recombinant baculovirus. SeeNawagitgul, P. et al., 2000, J. Gen. Virol. 81:2281-2287.

In some illustrative embodiments of the invention disclosed herein, arecombinant vector comprising a PCV genome or an ORF thereof, or aportion thereof, such as an antigenic region, is constructed by in vitrorecombination between a plasmid and a PCV genome. In some embodiments,the PCV genome is a PCV2 genome. In other embodiments, a recombinantvector comprising a PCV genome or an ORF thereof, or a portion thereof,such as an antigenic region, is constructed by in vivo recombination.Methods for in vivo recombination are know in the art and include, forexample, the methods disclosed in Chartier, et al. (1996, J. Virol.70:4805-4810). Vectors for constructing circovirus genomes include forexample, bacterial plasmids which allow multiple copies of the clonedcircovirus nucleotide sequence to be produced. In some embodiments, theplasmid is co-transfected into a suitable host cell for recombination.Suitable host cells for recombination include any cell that will supportrecombination between a PCV genome and a plasmid containing PCVsequences, or between two or more plasmids, each containing PCVsequences. Recombination is generally performed in procaryotic cells,such as E. coli for example, while generation of circovirus ispreferably performed in mammalian cells permissive for PCV replication,such as for example porcine cells and in particular, porcine cellscapable of expressing mammalian adenovirus E1 function.

The present invention encompasses the use of any mammalian host cellpermissive for circovirus replication, and in particular, permissive forreplication of PCV, such as PCV1 and PCV2. Allan et al. (1995,Veterinary Microbiology 44: 49-64) report that PCV replicate in porcineand bovine monocyte/macrophage cultures. Tischer et al. (1987, Arch.Virol. 96:39-57) report that PCV is known to require actively dividingcells for replication in cell culture. Examples of cells or cell linesuseful for replication of PCV include mammalian cells comprising E1function and permissive for PCV replication, including porcine cells,such as porcine monocyte/macrophage cells and porcine retinal cells,expressing adenovirus E1 function. In an illustrative embodimentdisclosed herein, porcine retina cells expressing human adenovirus E1function are shown to be permissive for replication of PCV2 and shown togenerate virus at 2×10⁷ IU/ml. Porcine cell lines are available frompublic sources such as for example, the American Type Tissue Collection(ATCC). The growth of bacterial cell cultures, as well as culture andmaintenance of eukaryotic cells and mammalian cell lines are procedureswell-known to those of skill in the art.

The present invention encompasses methods of culturing mammaliancircovirus, in particular, porcine circovirus, in mammalian host cellstransfected with mammalian adenovirus E1 gene sequences. In someembodiments, the mammalian cell is stably transformed with adenovirus E1gene sequences. In some embodiments, the E1 gene sequences areintegrated into the genome of the mammalian cell. In other embodiments,the E1 gene sequences are present on a replicating plasmid. In yet otherembodiments, the E1 gene sequence is heterologous to the mammalian cell.In an illustrative embodiment disclosed herein a porcine mammalian cellis transformed with a human adenovirus 5 E1 gene sequence. The presentinvention encompasses the use of any mammalian cell or mammalian cellline expressing E1 function as long as the mammalian cell or cell lineexpressing E1 function is permissive for the replication of circovirus,in particular porcine circovirus, such as for example, porcinecircovirus 1 or porcine circovirus 2. In preferred embodiments, themammalian cell is a porcine cell or cell line. The present inventionencompasses the use of any mammalian E1 function as long as themammalian host cell expressing the mammalian E1 function is permissivefor replication of circovirus, in particular, PCV, such as for example,porcine circovirus 1 or porcine circovirus 2. Mammalian adenovirusgenomes are known in the art and are disclosed in, for example, Reddy etal. (1998, Journal of Virology, 72:1394) which discloses nucleotidesequence, genome organization, and transcription map of bovineadenovirus 3 (BAV3); and Kleiboeker (1995, Virus Res. 36:259-268), whichdiscloses the E1 region of PAV-4. The present invention encompasses E1function from any of the various serotypes of human adenovirus, such asAd2, Ad5, Ad12, and Ad40. In an illustrative embodiment disclosed hereinin Example 1, E1 function is human Ad5 E1 function. The human E1A geneis expressed immediately after viral infection (0-2 hours) and beforeany other viral genes. Flint (1982) Biochem. Biophys. Acta 651:175-208;Flint (1986) Advances Virus Research 31:169-228; Grand (1987) Biochem.J. 241:25-38. The transcription start site of Ad5 E1A is at nucleotide498 and the ATG start site of the E1A protein is at nucleotide 560 inthe virus genome. The E1B protein functions in trans and is necessaryfor transport of late mRNA from the nucleus to the cytoplasm. The E1Bpromoter of Ad5 consists of a single high-affinity recognition site forSp1 and a TATA box. In particular, human adenovirus 5 E1A and E1B genesequences are located at nucleotides 505-4034 of the nucleotide sequenceprovided in Chroboezek, J. et al. (1992, Virology. 186:280-285). In anillustrative embodiment disclosed herein in the Examples, the mammalianhost cell is a porcine host cell transfected with human adenovirus 5 E1gene sequences.

The PCV genome can be isolated from PCV virions, or can comprise a PCVgenome that has been inserted into a plasmid, using standard techniquesof molecular biology and biotechnology. Cloning of the full-length PCV2genome into vector pBluescript II KS(+) from Strategene by PCR isdescribed in Liu, et al. (2000, J. Clin. Microbiol. vol 38:3474-3477).The full-length PCV2 genome DNA can be released from the resultingplasmid upon SacII digestion.

Introduction of circovirus nucleotide sequences into permissivemammalian host cells can be achieved by any method known in the art,including, but not limited to, transfection and transformationincluding, but not limited to, microinjection, electroporation, CaPO₄precipitation, DEAE-dextran, liposomes, particle bombardment, etc. Anillustrative method for transfecting PCV2 nucleotide sequences into VIDOR1 cells is described herein in Example 3.

Methods for culturing procaryotic cells, such as bacterial cells, andeukaryotic cells, such as mammalian host cells expressing adenovirus E1function are deemed routine to those of skill in the art.

The following examples are provided to illustrate but not limit theinvention. All references and patent publications disclosed herein arehereby incorporated in their entirety by reference.

EXAMPLES Example 1

Preparation of Porcine Retinal Cells Transfected with Human AdenovirusE1 Gene Sequences (VIDO R1 Cells)

Primary cultures of porcine embryonic retina cells were transfected with10 μg of plasmid pTG 4671 (Transgene, Strasbourg, France) by the calciumphosphate technique. The pTG 4671 plasmid contains the entire E1A andE1B sequences (nts 505-4034) of HAV-5, along with the puromycinacetyltransferase gene as a selectable marker. In this plasmid, the E1region is under the control of the constitutive promoter from the mousephosphoglycerate kinase gene, and the puromycin acetyltransferase geneis controlled by the constitutive SV40 early promoter. Transformed cellswere selected by three passages in medium containing 7 μg/ml puromycin,identified based on change in their morphology from single foci (i.e.,loss of contact inhibition), and subjected to single cell cloning. Theestablished cell line was first tested for its ability to support thegrowth of E1 deletion mutants of HAV-5. Subsequently the cell line wasfurther investigated for the presence of E1 sequences in the genome byPCR, expression of the E1A and E1B proteins by Western blot, anddoubling time under cell culture conditions. E1 sequences were detected,and production of E1A and E1B proteins was demonstrated byimmunoprecipitation. Doubling time was shorter, when compared to that ofthe parent cell line.

To assess the stability of E1 expression, VIDO R1 cells were culturedthrough more than 50 passages (split 1:3 twice weekly) and tested fortheir ability to support the replication of E1-deleted HAV-5. Expressionof the E1A and E1B proteins at regular intervals was also monitored byWestern blot. The results indicated that the VIDO R1 line retained theability to support the growth of E1-deleted virus and expressed similarlevels of E1 proteins during more than 50 passages in culture.Therefore, VIDO R1 can be considered to be an established cell line.VIDO R1 cell line has been deposited with the American Type Culturecollection (ATCC) and has ATCC accession number PTA-155.

Example 2

Example 2 provides a description of the molecular cloning of full-lengthPCV2 genome.

Initially, PCV2 DNA was amplified by PCR from total DNA extracted from apiglet with PMWS. The cloning of the full-length PCV2 genome DNA intovector pBluescript II KS(+) (Stratagene) by polymerase chain reaction(PCR) was described in Liu et al. (2000). J. Clin. Microbiol.38:3474-3477). The PCV2 sequence was submitted to GenBank (Accession no.AF086834). The full-length PCV2 genome DNA is released from theresulting plasmid upon SacII digestion.

Example 3

Example 3 describes the transfection of VIDO R1 cells, as described inExample 1, with a plasmid containing the PCV2 genome as constructed inExample 2.

Material and Methods

Cell Culture

Fetal porcine retina cell line, VIDO R1, as described in Example 1 andVero cells (ATCC) were maintained at 37° C. with 5% CO₂ in Eagles basedMEM media supplemented with 10% or 5% heat-inactivated fetal bovineserum (FBS), respectively.

Transfection and Infection

Monolayers of VIDO R1 cells grown in a six-well dish were transfectedwith cloned PCV2 DNA using Lipofectin according to the manufacturer'srecommendations (GIBCO BRL). Prior to transfection, PCV2 full-lengthgenome was released from the plasmid by digestion with SacII (Liu, Q.,et al., 2000, J. Clin. Microbiol. 38:3474-3477). For infection, thetransfected VIDO R1 cells were subjected to three cycles of freezing(−70° C.) and thawing (37° C.). The lysate was then clarified bycentrifugation and used to infect fresh VIDO R1 cells. In publishedreports, a PCV 1-free porcine kidney cell line is used to culture PCV2virus. To stimulate the entry to the S phase in the cell cycle, theporcine kidney cells are always treated by D-glucosamine (see Tischer etal., (1987). Arch Virol. 96:39-57.). However, the treatment must beperformed with caution because D-glucosamine is toxic for cell culture(see, Allan et al., (2000). J. Vet. Diagn. Investigation. 12:3-14). Incontrast, since the VIDO R1 cell line used in this study has beentransformed by HAV5-E1 that can induce the S phase, the D-glucosaminetreatment was not necessary.

Example 4

Virus Purification and Titration

For the purification of PCV2 virus, PCV2-infected VIDO R1 cells wereincubated with 0.5% Triton X-114 in phosphate-buffered saline (PBS) at37° C. for 45 min followed by Freon 113(1,1,2-trichloro-trifluoroethane) extraction. The cell debris andmembranes were clarified by centrifugation at 2000 g for 15 min. Theviruses in the supernatant were pelleted at 35000 g for 3 h through a20% sucrose cushion. The virus pellet was suspended in PBS and stored at−70° C. Virus titers were determined as infectious units (IU) byquantitative ORF2 protein immuno-peroxidase staining. For this purpose,the cell monolayers in 12-well dishes were infected with serialdilutions of virus. After adsorption of virus for 1 h, the cells werewashed and overlaid with MEM containing 2% FBS and 0.7% agarose. On day3 post infection (p.i.), the agarose overlay was removed and the cellswere fixed and permeabilized with methanol/acetone (1:1 in volume) for20 min at −20° C. After blocking with 1% bovine serum albumin for 1 h atroom temperature, the cells were incubated with rabbit anti-ORF2 serum(Liu et al., 2001, Protein Expression and Purification. 21:115-120).After 2 h incubation, the plates were washed with PBS and then processedusing VECTASTAIN Elite ABC kit (Vector Laboratories). The reaction wasdeveloped with 3,3′diaminobenzidine (DAB) tetrahydrochloride andobserved under a microscope. By counting the positively stained cells,the virus titer was expressed as IU where 1 IU was defined as onepositively stained cell/foci at 3 d.p.i.

Viral DNA Extraction and Characterization

Viral DNA was extracted from PCV2-infected VIDO R1 cell monolayers bythe method of Hirt (1967, J. Mol. Biol. 26:365-369). The viral DNA wasthen characterized by restriction analysis and polymerase chain reaction(PCR) as described in Liu et al., (2000). J. Clin. Microbiol.38:3474-3477).

PCR using DNA extracted from the infected cells as template andPCV-2-specific primers amplified a product of specific size, while noDNA was amplified from control, uninfected cells. Consistent with theexpected restriction patterns, digestions of viral DNA with NcoI andStuI resulted in two fragments of 1291 bp and 477 bp in size,respectively; digestion with EcoRI and StuI produced two fragments of1492 bp and 276 bp in size, respectively; and digestion with EcoRI andEcoRV generated two fragments of 1094 bp and 674 bp in size,respectively. The data indicate that PCV2 virus was obtained. Using animmunostaining assay and by counting the positive stained cells, thevirus titer of this preparation was determined to be 2×10⁷ IU/ml.

3 1 1768 DNA Porcine circovirus 1 accagcgcac ttcggcagcg gcagcacctcggcaacacct cagcagcaac atgcccagca 60 agaagaatgg aagaagcgga ccccaaccacataaaaggtg ggtgttcacg ctgaataatc 120 cttccgaaga cgagcgcaag aaaatacgggagctcccaat ctccctattt gattatttta 180 ttgttggcga ggagggtaat gaggaaggacgaacacctca cctccagggg ttcgctaatt 240 ttgtgaagaa gcaaactttt aataaagtgaagtggtattt gggtgcccgc tgccacatcg 300 agaaagccaa aggaactgat cagcagaataaagaatattg tagtaaagaa ggcaacttac 360 ttattgaatg tggagctcct cgatctcaaggacaacggag tgacctgtct actgctgtga 420 gtaccttgtt ggagagcggg attctggtgaccgttgcaaa gcagcaccct gtaacgtttg 480 tcaaaaattt ccgcgggctg gctgaacttttgaaagtgag cgggaaaatg caaaagcgtg 540 attggaaaac caatgtacac ttcattgtggggccacctgg gtgtggtaaa agcaaatggg 600 ctgctaattt tgcaaacccg gaaaccacatactggaaacc acctaaaaac aagtggtggg 660 atggttacca tggtgaaaaa gtggttgttattgatgactt ttatggctgg ctgccgtggg 720 atgatctact gagactgtgt gatcgatatccattgactgt aaaaactaaa ggtggaactg 780 tacctttttt ggcccgcagt attctgattaccagcaatca aaccccgttg gaatggtact 840 cctcaactgc tgtcccagct gtagaagctctctatcggag gattacttcc ttggtatttt 900 ggaagaatgt tacagaacaa tccacggaggaagggggcca gtttgtcacc ctttcccccc 960 catgccctga atttccatat gaaataaattactgagtctt ttttatcact tcgtaatggt 1020 ttttattatt catttagggt ttaagtggggggtctttaag attaaattct ctgaattgta 1080 catacatggt tacacggata ttgtagtcctggtcgtattt actgttttcg aacgcagtgc 1140 cgaggcctac gtggtccaca tttctagaggtttgtagcct cagccaaagc tgattccttt 1200 tgttatttgg ttggaagtaa tcaatagtggagtcaagaac aggtttgggt gtgaagtaac 1260 gggagtggta ggagaagggt tgggggattgtatggcggga ggagtagttt acatatgggt 1320 cataggttag ggctgtggcc tttgttacaaagttatcatc tagaataaca gcagtggagc 1380 ccactcccct atcaccctgg gtgatgggggagcagggcca gaattcaacc ttaacctttc 1440 ttattctgta gtattcaaag ggtatagagattttgttggt cccccctccc gggggaacaa 1500 agtcgtcaat attaaatctc atcatgtccaccgcccagga gggcgttgtg actgtggtag 1560 ccttgacagt atatccgaag gtgcgggagaggcgggtgtt gaagatgcca tttttccttc 1620 tccaacggta gcggtggcgg gggtggacgagccaggggcg gcggcggagg atctggccaa 1680 gatggctgcg ggggcggtgt cttcttctgcggtaacgcct ccttggatac gtcatagctg 1740 aaaacgaaag aagtgcgctg taagtatt1768 2 314 PRT Porcine circovirus 2 Met Pro Ser Lys Lys Asn Gly Arg SerGly Pro Gln Pro His Lys Arg 1 5 10 15 Trp Val Phe Thr Leu Asn Asn ProSer Glu Asp Glu Arg Lys Lys Ile 20 25 30 Arg Glu Leu Pro Ile Ser Leu PheAsp Tyr Phe Ile Val Gly Glu Glu 35 40 45 Gly Asn Glu Glu Gly Arg Thr ProHis Leu Gln Gly Phe Ala Asn Phe 50 55 60 Val Lys Lys Gln Thr Phe Asn LysVal Lys Trp Tyr Leu Gly Ala Arg 65 70 75 80 Cys His Ile Glu Lys Ala LysGly Thr Asp Gln Gln Asn Lys Glu Tyr 85 90 95 Cys Ser Lys Glu Gly Asn LeuLeu Ile Glu Cys Gly Ala Pro Arg Ser 100 105 110 Gln Gly Gln Arg Ser AspLeu Ser Thr Ala Val Ser Thr Leu Leu Glu 115 120 125 Ser Gly Ile Leu ValThr Val Ala Lys Gln His Pro Val Thr Phe Val 130 135 140 Lys Asn Phe ArgGly Leu Ala Glu Leu Leu Lys Val Ser Gly Lys Met 145 150 155 160 Gln LysArg Asp Trp Lys Thr Asn Val His Phe Ile Val Gly Pro Pro 165 170 175 GlyCys Gly Lys Ser Lys Trp Ala Ala Asn Phe Ala Asn Pro Glu Thr 180 185 190Thr Tyr Trp Lys Pro Pro Lys Asn Lys Trp Trp Asp Gly Tyr His Gly 195 200205 Glu Lys Val Val Val Ile Asp Asp Phe Tyr Gly Trp Leu Pro Trp Asp 210215 220 Asp Leu Leu Arg Leu Cys Asp Arg Tyr Pro Leu Thr Val Lys Thr Lys225 230 235 240 Gly Gly Thr Val Pro Phe Leu Ala Arg Ser Ile Leu Ile ThrSer Asn 245 250 255 Gln Thr Pro Leu Glu Trp Tyr Ser Ser Thr Ala Val ProAla Val Glu 260 265 270 Ala Leu Tyr Arg Arg Ile Thr Ser Leu Val Phe TrpLys Asn Val Thr 275 280 285 Glu Gln Ser Thr Glu Glu Gly Gly Gln Phe ValThr Leu Ser Pro Pro 290 295 300 Cys Pro Glu Phe Pro Tyr Glu Ile Asn Tyr305 310 3 233 PRT Porcine circovirus 3 Met Thr Tyr Pro Arg Arg Arg TyrArg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile LeuArg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg Tyr Arg Trp ArgArg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly TyrThr Val Lys Ala Thr Thr Val Thr Thr 50 55 60 Pro Ser Trp Ala Val Asp MetMet Arg Phe Asn Ile Asp Asp Phe Val 65 70 75 80 Pro Pro Gly Gly Gly ThrAsn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val LysVal Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg GlyVal Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Thr LysAla Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140 Ser Ser ArgHis Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 PheThr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn 180 185190 Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp 195200 205 Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe210 215 220 Asn Leu Lys Asp Pro Pro Leu Lys Pro 225 230

We claim:
 1. A method for culturing a mammalian circovirus comprising:a) obtaining mammalian cells expressing a mammalian adenovirus E1functional protein, wherein said cells are permissive for mammaliancircovirus replication; b) introducing the mammalian circovirus genome,or a portion thereof capable of replication, into said mammalian cells;and c) culturing said mammalian cells under conditions suitable forreplication of said mammalian circovirus.
 2. The method of claim 1further comprising recovering said circovirus from said cultured cells.3. The method of claim 1 wherein said mammalian circovirus is porcinecircovirus.
 4. The method of claim 3 wherein said porcine circovirus isporcine circovirus type
 2. 5. The method of claim 3 wherein said porcinecircovirus is porcine circovirus type
 1. 6. The method of claim 1wherein said mammalian cells are of porcine origin.
 7. The method ofclaim 1 wherein said mammalian cells are porcine retina cells.
 8. Themethod of claim 1 wherein said mammalian adenovirus E1 functionalprotein is human adenovirus E1 functional protein.
 9. The method ofclaim 1 wherein said mammalian adenovirus E1 functional protein isporcine adenovirus E1 functional protein.
 10. The method of claim 1wherein said mammalian cells expressing the mammalian adenovirus E1functional protein are stably transformed with a mammalian adenovirus E1gene sequence.
 11. The method of claim 10 wherein said E1 gene sequenceis a human adenovirus E1 gene sequence.
 12. The method of claim 10wherein said mammalian adenovirus E1 gene sequence is heterologous tosaid mammalian cell.
 13. The method of claim 1 wherein said E1functional protein is E1A and/or E1B functional protein.
 14. The methodof claim 3 wherein said porcine circovirus comprises a chimericnucleotide sequence.
 15. A method of preparing a recombinant mammaliancell expressing a mammalian adenovirus E1 functional protein andcomprising a porcine circovirus genome comprising the steps of, a)obtaining a mammalian cell expressing a mammalian adenovirus E1functional protein; and b) introducing said porcine circovirus genome,or a portion thereof capable of replication, into the mammalian cell.16. The method of claim 15 further comprising the step of culturing saidrecombinant mammalian cell under conditions suitable for the replicationof said porcine circovirus.
 17. The method of claim 16 furthercomprising recovering said circovirus from said cultured cells.
 18. Themethod of claim 15 wherein said porcine circovirus is porcine circovirustype
 2. 19. The method of claim 15 wherein said porcine circovirus isporcine circovirus type
 1. 20. The method of claim 15 wherein saidmammalian cells are of porcine origin.
 21. The method of claim 20wherein said mammalian cells are porcine retina cells.
 22. The method ofclaim 15 wherein said adenovirus E1 functional protein is humanadenovirus E1 functional protein.
 23. The method of claim 15 whereinsaid adenovirus E1 functional protein is porcine adenovirus E1functional protein.
 24. The method of claim 15 wherein said porcinecircovirus comprises a chimeric nucleotide sequence.
 25. The method ofclaim 15 wherein said mammalian cell expressing a mammalian adenovirusE1 functional protein is stably transformed with a mammalian adenovirusE1 gene sequence.
 26. The method of claim 25 wherein said mammalianadenovirus E1 gene sequence is heterologous to said mammalian cell. 27.A method for replicating a mammalian circovirus comprising, culturing amammalian cell that comprises a mammalian circovirus genome, or aportion thereof capable of replication, under conditions suitable forreplication of said mammalian circovirus, wherein the mammalian cellexpresses a mammalian adenovirus E1 functional protein and is permissivefor mammalian circovirus replication, and optionally recovering saidcircovirus from the cultured cell.
 28. The method of claim 27 whereinsaid mammalian circovirus is porcine circovirus.
 29. The method of claim28 wherein said porcine circovirus is porcine circovirus type
 2. 30. Themethod of claim 28 wherein said porcine circovirus is porcine circovirustype
 1. 31. The method of claim 27 wherein said mammalian cells are ofporcine origin.
 32. The method of claim 31 wherein said mammalian cellsare porcine retina cells.
 33. The method of claim 27 wherein saidmammalian adenovirus E1 functional protein is human adenovirus E1functional protein.
 34. The method of claim 27 wherein said mammalianadenovirus E1 functional protein is porcine adenovirus E1 functionalprotein.
 35. The method of claim 27 wherein said mammalian cellsexpressing the mammalian adenovirus E1 functional protein are stablytransformed with a mammalian adenovirus E1 gene sequence.
 36. The methodof claim 35 wherein said E1 gene sequence is a human adenovirus E1 genesequence.
 37. The method of claim 35 wherein said mammalian adenovirusE1 gene sequence is heterologous to said mammalian cell.
 38. The methodof claim 27 wherein said E1 functional protein is E1A and/or E1Bfunctional protein.
 39. The method of claim 28 wherein said porcinecircovirus comprises a chimeric nucleotide sequence.
 40. A method ofpreparing a recombinant mammalian cell comprising, introducing amammalian circovirus genome, or a portion thereof capable ofreplication, into a mammalian cell that expresses a mammalian adenovirusE1 functional protein, wherein the cell is permissive for replication ofthe mammalian circovirus.
 41. A method of preparing a recombinantmammalian cell comprising, introducing a mammalian adenovirus E1 generegion into a mammalian cell that comprises a mammalian circovirusgenome, or a portion thereof capable of replication, wherein the cell ispermissive for replication of the mammalian circovirus.
 42. The methodof claim 40 or 41 further comprising the step of culturing saidrecombinant mammalian cell under conditions suitable for the replicationof said circovirus.
 43. The method of claim 42 further comprisingrecovering said circovirus from said cultured cell.
 44. The method ofclaim 40 or 41 wherein said circovirus is porcine circovirus.
 45. Themethod of claim 44 wherein said porcine circovirus is porcine circovirustype 1 or porcine circovirus type
 2. 46. The method of claim 40 or 41wherein said mammalian cell is of porcine origin.
 47. The method ofclaim 46 wherein said mammalian cell is a porcine retina cell.
 48. Themethod of claim 40 or 41 wherein said adenovirus E1 functional proteinis human adenovirus E1 functional protein.
 49. The method of claim 40 or41 wherein said adenovirus E1 functional protein is porcine adenovirusE1 functional protein.
 50. The method of claim 40 or 41 wherein saidcircovirus comprises a chimeric nucleotide sequence.
 51. The method ofclaim 40 or 41 wherein said mammalian cell is stably transformed with amammalian adenovirus E1 gene sequence.
 52. The method of claim 40 or 41wherein said mammalian adenovirus E1 gene sequence is heterologous tosaid mammalian cell.
 53. The method of claim 40 or 41 wherein said E1functional protein is E1A and/or E1B functional protein.
 54. A methodfor expressing a mammalian circovirus genome, or portion thereof,comprising, culturing a mammalian cell that comprises the mammaliancircovirus genome, or a portion thereof, under conditions suitable forexpression of said mammalian circovirus genome, or portion thereof,wherein the mammalian cell expresses a mammalian adenovirus E1functional protein and optionally recovering said circovirus, or portionthereof, from the cultured cell.
 55. The method of claim 54 wherein saidmammalian circovirus is porcine circovirus.
 56. The method of claim 55wherein said porcine circovirus is porcine circovirus type
 2. 57. Themethod of claim 55 wherein said porcine circovirus is porcine circovirustype
 1. 58. The method of claim 54 wherein said mammalian cell is ofporcine origin.
 59. The method of claim 58 wherein said mammalian cellis a porcine retina cell.
 60. The method of claim 54 wherein saidmammalian adenovirus E1 functional protein is human adenovirus E1functional protein.
 61. The method of claim 54 wherein said mammalianadenovirus E1 functional protein is porcine adenovirus E1 functionalprotein.
 62. The method of claim 54 wherein said mammalian cell isstably transformed with a mammalian adenovirus E1 gene sequence.
 63. Themethod of claim 62 wherein said E1 gene sequence is a human adenovirusE1 gene sequence.
 64. The method of claim 62 wherein said mammalianadenovirus E1 gene sequence is heterologous to said mammalian cell. 65.The method of claim 15 or 54 wherein said E1 functional protein is E1Aand/or E1B functional protein.
 66. The method of claim 54 wherein saidporcine circovirus comprises a chimeric nucleotide sequence.